Technical Abstract: Changes in apoplast carbohydrate concentrations and activities of carbohydrate degrading enzymes were determined in crown tissues of oat (Avena sativa, L., cv. Wintok) during cold hardening. During second phase hardening (-3 degrees C for 3 days) crown apoplast levels of sucrose, glucose and fructose increased from 0.9%, 2.1%, and 2.0%, respectively, of whole tissue levels in first phase hardened plants (3 degrees C for 3 weeks) to 6.4%, 17.6% and 14.9%, respectively. Fructan in the crown apoplast also increased from 0.5% to 0.8% in non-hardened and first phase hardened plants to 2.4% to 7.9% in second phase hardened plants. The extent of increase in apoplast fructan during second phase hardening varied with degree of fructan polymerization (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP>7). Activities of invertase and fructan exohydrolase in crown apoplast increased ca. 4-fold over non-hardened and first phase hardened plants to 8.3% and 11.9%, respectively, of whole tissue activity. Apoplastic fluid extracted from non-hardened, first phase hardened and second phase hardened crown tissues had low levels (0.7, 1.1, and 2.8%, respectively) of symplastic contamination as determined by malate dehydrogenase activity. The significance of these results in relation to previously reported increases freezing tolerance from second phase hardening is discussed.